The proposed research is a continued study of the involvement of proteins in splicing group I and group II introns. These introns use RNA-catalyzed splicing mechanisms, but require proteins for efficient splicing in vivo to help fold the intron RNA into the catalytically active structure. We showed previously that the Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) recognizes conserved tRNA-like structural features of the group I intron catalytic core and is by itself sufficient to promote the splicing of different group I introns in vitro. During the current grant period, we obtained a 2.5Angstrom crystal structure of a splicing-compete CYT-18 protein and identified a DEAD-box protein (CYT-19) that functions in concert with CYT-18 to promote group I intron splicing by acting as an ATPdependent RNA chaperone. In the proposed research, we would continue structural analysis of CYT-18 and its complexes with group I intron RNAs and use CYT-19 as a model system for studying how DExH/D-box proteins mediate RNA conformational changes and acquire specificity for their target RNAs. For group II introns, we have developed an experimental system centered around the mobile Lactococcus lactis L1.LtrB intron, which encodes a reverse transcriptase (LtrA protein) that functions in mobility and as an intron-specific splicing factor ("maturase"). Studies during the current grant period have led to a model in which the LtrA protein promotes splicing by binding first to a high affinity binding site in intron subdomain DIVa, an idiosyncratic structure at the beginning of the LtrA coding region, and then makes additional contacts with conserved regions of the catalytic core to fold the RNA into the catalytically active structure. In the proposed research, we would continue to study the mechanism of maturase-promoted group II intron splicing, explore the involvement of DExH/D-box proteins in group II intron splicing, and investigate whether maturases can evolve into general group II intron splicing factors, potentially mirroring a key step in the evolution of spliceosomal introns. Specific aims are: (1) To continue structural analysis of the N. crassa CYT-18 protein and its complexes with group I intron RNAs. (2) To continue to investigate how the DEAD-box protein CYT- 19 functions in group I intron splicing and its targeting to CYT-18-dependent group I introns. (3) To continue to investigate the mechanism of maturase-promoted group II intron splicing. (4) To explore the role of DExH/D-box proteins in group II intron splicing. (5) To explore whether some maturases, including chloroplast MatK proteins, can evolve to function in splicing multiple group II introns. This research is intended to provide novel information about how proteins mediate RNA folding and RNA-catalyzed reactions, as well as insight into the evolution of introns and splicing mechanisms, which are fundamentally important for gene expression in higher organisms.